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Postprint of LWT - Food Science and Technology V. 66, 2016, Pages 378-383 1

DOI: https://doi.org/10.1016/j.lwt.2015.10.063 2

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Fatty Acid Ethyl Esters (FAEE) in extra virgin olive oil: a case study of a 4

quality parameter1. 5

Raquel B. Gómez-Cocaa,*, Gabriel D. Fernandesb, María del Carmen Pérez-6

Caminoa, and Wenceslao Moredaa 7

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aDepartment of Characterization and Quality of Lipids. Instituto de la Grasa –9

CSIC-, Campus of Universidad Pablo de Olavide, Building 46, Ctra. de Utrera, 10

km 1, 41013 Sevilla, Spain. 11

bFat and Oil Laboratory, Faculty of Food Engineering, University of Campinas, 12

13083-970 Campinas, SP, Brazil 13

*Corresponding author. 14

E-mail address: raquel.coca@ig.csic.es (R. B. Gómez-Coca) 15

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ABSTRACT 18

1 C16 ET, C16 fatty acid ethyl esters; C16 ME, C16 fatty acid methyl esters; C17:0 ME, methyl heptadecanoate; C18:1, oleic acid; C18 ET, C18 fatty acid ethyl esters; C18 ME, C18 fatty acid methyl esters; EtOH, ethanol; EVOO, extra virgin olive oil; FAAE, fatty acid alkyl esters; FAEE, fatty acid ethyl esters, FAME, fatty acid methyl esters; FID, flame ionization detector; GC, gas chromatography; IOC, International Olive Council; IS, Internal Standard; MeOH, methanol; PrOH, 1-propanol; PTFE, polytetrafluoroethylene; SDr, standard deviation of the repeatability.

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After establishing the relationship between fatty acid alkyl esters (FAAE) in olive 19

oil and its sensory classification, we proved the correlation between the 20

presence of large quantities of FAAE and the oil’s fermentative defects. 21

Nowadays the olive oil industry is facing strict demands regarding the fatty 22

acid ethyl ester (FAEE) presence in extra virgin olive oil, since a 30 mg/kg limit 23

must be applied to oils produced from 1st March 2016. This decision was made 24

under the assumption that the concentration of FAEE is something fixed. 25

Results here demonstrate otherwise. After a study under controlled storage 26

conditions (temperature, free acidity and volatiles), it is shown that the FAEE 27

concentration increases dramatically over time once the oil is bottled. This, in 28

the case of extra virgin olive oils obtained from mature healthy fruits, may lead 29

in a few month time to FAEE concentrations above the limit permitted to classify 30

the oils as extra virgin, underlying the need of applying certain working practices 31

systematically such as filtering prior bottling, and strict control of the storage 32

temperature. 33

34

Keywords: acidity, FAAE, FAME, filtration, volatiles. 35

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37

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1. Introduction 39

According to the lexicon, to characterize means to present or to describe 40

something through its distinguishing features. It is widely accepted in the olive 41

oil world that the special and typical organoleptic profile of EVOO is the one that 42

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will differentiate it from the rest of the oils, including olive oils from other 43

categories. 44

Olive oil organoleptic assessment plays a relevant role in olive oil 45

classification since it is included in the quality parameters required to allocate a 46

certain oil within one of the legally recognized olive oil categories (European 47

Commission Regulation, 1991). In this case the organoleptic evaluation is made 48

by a trained panel of experts (International Olive Council, 2011a; International 49

Olive Council, 2011b) where oil’s rejection is not a question of acceptance –as 50

in the case of, e.g., preference tests focused on market research (Angerosa & 51

Campestre, 2013)- but a more objective issue. Expert tasters will score 52

positively features such as bitterness, pungency or fruitiness, whereas attributes 53

such as musty, winey-vinegary or muddy sediment will be considered as 54

defects present in the oil due to the utilization of low quality fruits in which 55

fermentative processes have occurred (European Commission Regulation, 56

1991). There also exist an intermediate and above everything illicit situation in 57

which poor quality virgin olive oils with low organoleptic defects and poor market 58

value (but that would be perfectly accepted by most consumers) are subjected 59

to, e.g., soft deodorization followed by blending with EVOO, in order to mask 60

their negative flavour in front of a panel of experts and therefore to enhance its 61

market price. This is difficult to detect and so far analytical approaches have not 62

been successful enough to unmistakably differentiate this kind of fraud (Serani 63

& Piacenti, 2001; Serani, Piacenti, & Staiano 2001; Saba, Mazzini, Riffaelli, 64

Mattei, & Salvadori, 2005). To overcome this situation the determination of the 65

content the FAAE was proposed since it was demonstrated that they were 66

present at a certain concentration when olive fruits with fermentative alterations 67

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had been used for oil extraction (Pérez-Camino,Cert, Romero-Segura, Cert-68

Trujillo, & Moreda, 2008; Bendini, Cerretani, Valli, Lercker, & Mazzini, 2009). In 69

fact a relationship between the presence of large quantities of FAAE and the 70

sensory classification was established (Gómez-Coca, Moreda, & Pérez-71

Camino, 2012). 72

In past years Authorities introduced the FAAE determination as quality 73

parameter that would directly differentiate between extra virgin and non-extra 74

virgin olive oils. This would assure the maximum quality from the point of oil 75

extraction to that of oil bottling. In this way, one had to report the sum of the 76

contents of the FAME and the FAEE from C16 to C18 fatty acids and the total of 77

the two. The limit was set at 75 mg/kg but higher concentrations were allowed 78

provided that they did not exceed 150 mg/kg and that the FAEE/FAME ratio 79

was 1.5 at the maximum (International Olive Council, 2010; European 80

Commission Regulation, 2011). 81

The knowledge that EtOH was produced as metabolic by-product after 82

alcoholic fermentation (Conte, Mariani, Gallina Toschi, & Tagliabue, 2014) 83

drove to conclude that the presence of high concentration of both FAEE and 84

EtOH would evidence the use of, e.g., fermented olive fruits for oil extraction. 85

Therefore, new requirements were officially published. According to those only 86

C16 ET and C18 ET were to be taken into account in order to decide if a certain 87

olive oil could be classified as extra virgin. This decision was accompanied by a 88

reduction of the maximum allowed limit to 40 mg/kg (2013-14 crop year). 89

Additionally, it was approved to decrease such threshold by 5 mg/kg per year 90

within the two subsequent years (European Commission Regulation, 2013; 91

International Olive Council, 2013a) even considering a further 25 mg/kg 92

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threshold (Olimerca, 2015). These reductions represent undoubtedly a critical, 93

although not new, situation since some virgin olive oils first declare as extra 94

virgin will be classified as non-extra virgin. It is clear then clear: That the FAEE 95

content determined after oil extraction will be conditioned by the maturity index 96

of the olive fruit from which it had been extracted, being really high if poor 97

quality, overripe, fermented fruits have been used; that both fruit characteristics 98

and good manufacturing practices become crucial for oil quality (Biedermann, 99

Bongartz, Mariani, & Grob, 2008; Mariani, & Bellah, 2011); and that some parts 100

of the olive oil obtaining process (e.g. filtering prior bottling) may have to be 101

further optimized. However, worries may arise regarding the FAEE suitability as 102

quality parameter when taking into account the following facts: First of all, it has 103

been demonstrated that ethanol is not only a fermentation by-product, but that it 104

also accumulates in perfectly healthy fruits during their maturation on the tree 105

(Beltrán, Bejaoui, Jimenez, & Sanchez-Ortiz, 2015) as derivative in reactions 106

directed to produce, e.g., aroma compounds (Pesis, 2005). Secondly, it has 107

been proven that certain technological processes, e.g., water addition during 108

the oil extraction procedure, can change the original EtOH concentration in the 109

oil and therefore FAEE formation (Olimerca, 2015). Finally, with the decreasing 110

limits for FAEE concentration, worries arose regarding oil’s behaviour during 111

storage, since the slightest FAEE formation after oil extraction when using 112

healthy, mature fruits may push EVOO out of the extra virgin classification. So, 113

taking into account the importance of FAEE as quality parameter and in order to 114

increase our knowledge on this subject, we decided to check if EVOO would still 115

remain in the 30 mg/kg legal limit (European Commission Regulation, 2013; 116

International Olive Council, 2013a) after a certain time. Therefore, we carried 117

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out a study under controlled conditions (storage temperature and substrate 118

availability), measuring FAAE (FAME and FAEE) concentrations from the 119

moment of the extraction on. We then studied the ester formation in 120

dependence on the substrate availability (free fatty acids and short-chain 121

alcohols such as EtOH and MeOH). 122

The goal of this work is to study how good quality virgin olive oil (a product of 123

wide scope and significance in the food market) behaves with respect to FAEE 124

formation within the frame of the reduction of the maximum allowed limits. 125

Needless to say that there will be other aspects of the oil, e.g., oxidative quality 126

bound to polyphenol composition, etc., that are also affected by the 127

experimental conditions, but such study is beyond the scope of this article. 128

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2. Materials and methods 132

2.1 Chemicals 133

All chemical reagents were of recognized analytical quality. Water was either 134

distilled or of equivalent purity. The standards of C17:0 ME and C18:1, 135

phenolphthalein, potassium hydroxide, silica gel, 60-200 μm mesh, and Sudan I 136

(1-phenylazo-2-naphthol) were purchased from Sigma (St. Louis, MO, USA). 137

EtOH, ethyl ether, n-hexane, n-heptane, MeOH and PrOH were from Romil Ltd. 138

(Waterbeach, Cambridge, GB). 139

140

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2.2 Samples 142

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A set of 45 EVOO bottles was provided directly by producers from different 143

geographical origins in Andalucía, Spain. These oils were referred as coupage 144

oils, meaning they were the result of the fine mixing between each producer’s 145

varieties. 146

These samples were classified as belonging to high quality EVOO by the 147

Official Panel of Tasters of the Instituto de la Grasa (CSIC) in Seville, Spain 148

(International Olive Council, 2013b), as previously described (Gómez-Coca, 149

Moreda, & Pérez-Camino, 2012). 150

151

152

2.3 Sample preparation 153

In order to assure that there would be enough quantity of identical oils 154

through out the study, an only starting blend was prepared by gradually adding 155

increasing amounts from all 45 oils and mixing by magnetic stirrers. An alkyl 156

ester analysis was performed (International Olive Council, 2012) to make sure 157

that there were no chromatographic peaks within the retention time windows of 158

the FAAE under study. Also the free acidity expressed as percentage of oleic 159

acid (European Commission Regulation, 1991), and the presence of EtOH and 160

MeOH (Gómez-Coca, Cruz-Hidalgo, Fernandes, Pérez-Camino, & Moreda, 161

2014) were checked. This starting sample was divided in two portions, one of 162

them to be kept at room temperature (20 ºC) and the other at 40 ºC. From each 163

portion a set of batches was prepared dividing each of them into three aliquots 164

and spiking them with C18:1 till the free acidity was around 0.2, 0.4, and 0.7 %, 165

respectively. Subsequently, each of these aliquots was once again distributed 166

into three equal portions, which were spiked with EtOH and MeOH at 167

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concentrations of approximately 20, 40 and 60 mg/kg, respectively. All samples 168

were permanently protected from light. According to the described procedure 169

each alkyl ester determination (FAME, FAEE, and FAAE) encompassed a total 170

of 18 measurements made, at least, in duplicate. This experimental approach 171

has the advantage of including real temperature conditions mimicking both a 172

good storage situation and a less optimal circumstance in which samples are 173

subjected to high temperature. The fact of spiking the EVOO samples with 174

MeOH, EtOH, and C18:1 (see Section 2.3) instead of using virgin olive oil 175

samples of different qualities, obeys to the goal of starting from a olive oil matrix 176

identical in all cases, since it would be unpractical to measure the same analyte 177

in systems with different initial background composition. The chosen volatile 178

concentrations (20, 40, and 60 mg/kg) correspond to the standard concentration 179

found in EVOO, a higher concentration measured in some single-variety oils 180

(data not shown), and the concentration mimicking accelerated experimental 181

conditions, respectively. 182

183

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2.4 Analysis of fatty acid alkyl esters (FAAE) 185

Stock solutions of C17:0 ME were prepared by dissolving this standard in n-186

heptane at a concentration of 5 mg/L. 187

Sudan I dye was prepared at 1 mg/mL in a solution of n-hexane in ethyl ether 188

at 990 mL/L. 189

Samples were prepared just before the analysis. They consisted of a mixture 190

of 0.1 (± 0.001) g oil and 1 mL C17:0 ME solution, utilized as IS; 100 μL of the 191

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Sudan I solution were also added to check visually that the analytes were 192

eluted properly. 193

A procedure recommended by the IOC was used (International Olive 194

Council, 2012). In short: 3 g of silica gel suspended in n-hexane were 195

introduced into the chromatographic column and made to settle homogenously. 196

The silica was then conditioned with 10 mL n-hexane. Thereafter, the sample 197

prepared as described above was transferred onto the column followed by two 198

1 mL n-hexane rinses. Washing was made with 10 mL n-hexane. The adsorbed 199

esters were then eluted with 30 mL of a freshly prepared solution of n-hexane in 200

ethyl ether at 990 mL/L. The eluate was evaporated in a rotary evaporator at 201

room temperature under vacuum until a volume of 2 mL, which was then dried 202

under a gentle nitrogen flux, dissolved in 0.5 mL n-heptane, and analysed by 203

GC as described in Section 2.7. 204

205

206

2.5 Peak identification and quantitative analysis 207

The methyl and ethyl esters of the principal fatty acids found in olive oil (C16 208

ME, C16 ET, C18 ME and C18 ET, respectively) were identified following 209

published information (Pérez-Camino, Moreda, Mateos, & Cert, 2002). 210

The quantification of each peak was carried out on the basis of the area 211

corresponding to the C17:0 ME IS as described previously (Gómez-Coca, 212

Moreda, & Pérez-Camino, 2012). The results were reported as the sum of the 213

content of the methyl and ethyl esters from C16 to C18, and the total of the two, 214

expressed to the nearest mg/kg. 215

216

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217

2.6 Analysis of volatiles: ethanol and methanol 218

The determination of EtOH and MeOH was carried out according to a 219

published procedure (Gómez-Coca, Cruz-Hidalgo, Fernandes, Pérez-Camino, 220

& Moreda, 2014). Summarizing: concentrated PrOH (IS) solutions were 221

prepared in refined olive-pomace oil (2.5 mL/kg). From these concentrated 222

solutions, diluted solutions were made by mixing 1 g concentrated solution with 223

24 g refined olive-pomace oil. 224

Samples were prepared just before the analysis in the following way: 3.00 g 225

oil together with 300 mg diluted IS solutions were introduced into a 9 mL vial, 226

which was immediately sealed. They were heated in a dry heat bath at 110 ºC 227

during 60 min. The vial headspace was then sampled via a thermostated 228

stainless steel syringe (110 ºC; sampling time = 30 s) and analysed by GC (see 229

Section 2.7). 230

231

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2.7 Instrumentation 233

GC analyses of the FAAE were carried out with an Agilent 6890N Gas 234

Chromatograph (Agilent Technologies, Santa Clara, California, USA) as 235

described in previous publications (Gómez-Coca, Moreda, & Pérez-Camino, 236

2012), although with some modifications. In this sense, conditions for the GC 237

assays were: HP-5 fused silica capillary column (5 % diphenyl-95 % 238

dimethylpolysiloxane; 15 m, 0.32 mm ID, 0.10 μm film; Agilent Technologies, 239

Santa Clara, California, USA), 2.0 μL injection volume, hydrogen carrier gas at 240

9.6 mL/min and ECP cool on-column injection. The oven temperature program 241

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was: 70 ºC, rise at 10 ºC/min to 180 ºC, then at 5 ºC/min to 220 ºC, and finally 242

at 10 ºC/min to 340 ºC, 10 min. The detector temperature was 350 ºC. 243

GC analyses of the volatiles were done with an Agilent 7890B Gas 244

Chromatograph equipped with a Tracer MHS123 2t® Head Space Sampler and 245

FID (Agilent Technologies, Santa Clara, California, USA), as described 246

somewhere else (Gómez-Coca, Cruz-Hidalgo, Fernandes, Pérez-Camino, & 247

Moreda, 2014). 248

249

250

2.8 Determination of free fatty acids. 251

The determination of the free fatty acids expressed as the percentage of 252

oleic acid was carried out after the procedure published by the European 253

Commission (European Commission Regulation, 1991). According to this, 254

samples were dissolved in a mixture of equal parts by volume of diethyl ether 255

and ethanol (950 mL/L), and titrated using a titrated 0.1 mol/L potassium 256

hydroxide (56.11 g/mol) ethanolic solution, using phenolphthalein as indicator. 257

The acidity was expressed as a percentage by weight and the result as the 258

arithmetic mean of two calculations. Oleic acid molar weight (282 g/mol) was 259

used in the calculations since this is the acid utilized to express results. 260

261

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3. Results and discussion 264

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FAEE concentration is a quality parameter that reflects fruit quality at the 265

moment of the extraction. The evolution of this and other quality parameters are 266

independent from each other and occur in different ways. 267

In this study olive oil samples have been spiked with short-chain alcohols 268

and free oleic acid; furthermore, half of them have been storage at 40 ºC. It is 269

clear that not only the original FAAE content was going to be altered, but also 270

that other quality parameters such as the peroxide content or the UV absorption 271

were going to suffer some transformation, probably reaching limits incompatible 272

with high quality EVOO. However, the development of a comprehensive quality 273

study is quite beyond the scope of this paper and therefore is not going to be 274

discussed in this work, whose only focus is FAAE as the sum of FAME and 275

FAEE. 276

FAAE are formed by esterification of free fatty acids with short chain 277

alcohols, mainly MeOH and EtOH, yielding methyl and ethyl esters, 278

respectively. FAAE formation takes place easily in acid medium. The 279

development of this first order reaction depends on both temperature and 280

substrate presence. Therefore, we have followed the oil’s behaviour at both 281

normal storage conditions (around 20 ºC, room temperature) and somehow 282

more extreme situation (40 ºC, accelerated conditions), at acidity values 283

according to which the oils would be classified as extra virgin –although they 284

wouldn’t be of the same quality-, and with volatile content much higher than that 285

typically found in EVOO of the highest quality (Mariani, & Bellan, 2012; Gómez-286

Coca, Cruz-Hidalgo, Fernandes, Pérez-Camino, & Moreda, 2014). 287

288

289

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3.1 Analysis of fatty acid methyl and ethyl esters: product formation 290

The selected chromatographic conditions lead to the separation of the 291

individual esters according to the number of carbon atoms. In this way the gas 292

chromatograms consist of five peaks whose retention times appear within the 293

range from 8.0 to 10.3 min, corresponding to C16 ME, C16 ET, C17:0 ME (IS), 294

C18 ME, and C18 ET. 295

Generally speaking (for every acidity degree and every volatile 296

concentration) it has been observed that, according to the reaction kinetic, the 297

higher the temperature, the higher the ester formation, regardless if one 298

considers the methyl (data not shown) or ethyl ester contents (Table 1) 299

separately, or the total alkyl ester concentration (Fig. 1). This is because the 300

high temperature increases the proportion of reactant molecules (substrates) 301

whose energy is higher than the activation energy, giving rise to a higher 302

concentration of products at a certain time. 303

Since the limit presently into effect only takes into account the FAEE 304

presence, the discussion has been focused on the ethyl ester (C16 ET and C18 305

ET) formation, although the results are quite similar for all products and a 306

parallel reasoning may be followed in the case of, e.g., the FAAE (Fig. 1) 307

Table 1 shows the numerical results for the different conditions tested. The 308

first third of this table gives the figures corresponding to the lowest acidity value 309

(0.22 %). As expected, the higher the substrate concentration (EtOH), the 310

greater the product formation (FAEE). At the lowest EtOH concentration (24 311

mg/kg) all the samples kept at 20 ºC remained within the legal range (that is, 312

with a FAEE content below 30 mg/kg, which is the one required for the oil to be 313

considered as extra virgin) during the time of the measurements. However, 314

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those at 40 ºC presented FAEE concentrations above 30 mg/kg after seven and 315

a half months. Actually, the moment that the presence of EtOH was more 316

important (around 43 mg/kg) the temperature seemed to have a dramatic effect 317

and samples kept at 40 ºC were in around five-month time out of limit. 318

The second third of Table 1 shows homologous results at higher acidity (0.38 319

%). Again, substrate availability enhances product formation. Attention is to be 320

paid even at the lowest volatile concentration, since the samples kept at 40 ºC 321

were above the FAEE maximum allowed limit before five months; this period 322

was reduced to two and a half months at the highest EtOH concentration. 323

However, when the storage conditions were optimal (20 ºC), even at the highest 324

volatile concentrations, the 30 mg/kg limit was not exceeded. 325

Finally, in the case of oils with an acidity level near the 0.8 % threshold (0.77 326

%), the only samples that at the end of the study (around eight months) clearly 327

showed a FAEE concentration below the 30 mg/kg were those with a relatively 328

low EtOH presence, provided that they were not exposed at high temperatures 329

(third part of Table 1). 330

331

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3.2 Free acidity and volatile concentration: substrate availability 333

When we looked at how the substrate presence evolved, we verified that at 334

20 ºC there was a reduction of both, free acidity (from 0.22 to 0.18 %, from 0.38 335

to 0.36 %, and from 0.77 to 0.53 %) and EtOH concentration (decreases 336

between 29 and 49 %), which made sense since substrates were disappearing 337

when products were formed. A similar behaviour was observed at 40 ºC (EtOH 338

concentration diminished between 33 and 65 %) except for the fact that even if 339

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free fatty acids were being consumed due to FAEE formation, triglyceride 340

hydrolysis was strong enough to raise the acidity value progressively (from 0.22 341

to 0.41 %, from 0.38 to 0.73 %, and from 0.77 to 0.92 %), revealing once again 342

the importance of controlling the storage temperature. The law of mass action 343

states that the speed of a chemical reaction is proportional to the quantity of the 344

reacting substances, therefore the boosted FAEE formation at 40 ºC when 345

compare with the same situation at 20 ºC. 346

The importance of the temperature on the presence of free fatty acid and 347

therefore on the FAEE formation is also supported by the observations made at 348

different acidity values when comparing results from approximately the same 349

EtOH concentration. 350

Consequently, at high temperature at least two factors must be considered to 351

have an influence over the increase in FAEE formation in comparison with that 352

at room temperature: A) The enlarged number of reacting molecules charged 353

with enough energy as to surpass the activation energy of the reaction. B) The 354

enhanced triglyceride hydrolysis, which provides the media with higher 355

substrate concentration. 356

As far as the presumptive EtOH formation is concern, oil filtering before 357

storage prevents the presence of water within the oil matrix, therefore possible 358

fermentation reactions. That would explain the fact that the EtOH present in the 359

media is just consumed and not produced. It is also important to point out that 360

aqueous media will also enhance the presence of alcohols since it will act as 361

media for their solution. 362

363

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365

4. Final Remarks and Conclusions 366

Olive oil is a very complex food bound to a strong industry whose current 367

world production reaches easily three million tons of oil per year (Vossen, 2013) 368

therefore, any decision regarding the corresponding trade standards must be 369

founded on objective data and not be biased by the desires of the different 370

parties involved. 371

This work has focused on how virgin olive oil behaves with respect to FAAE 372

formation within the frame of the reduction of the maximum allowed limits from 373

75 mg/kg (FAAE) to 30 mg/kg (FAEE) (European Commission Regulation, 374

2013; International Olive Council, 2013a). 375

We have shown that the presence of a certain amount of FAEE is not always 376

indicative of poor quality. EtOH and FAEE will always be present in newly 377

extracted oils classified as extra virgin since it was demonstrated that EtOH is 378

not only a fermentation by-product but that it is also formed in the fruit during 379

aroma development (Beltrán, Bejaoui, Jimenez, & Sanchez-Ortiz, 2015). Both 380

EtOH and FAEE concentrations in oil will be low if fruits with low maturity index 381

have been used, or somehow higher in the case of oil from mature olives. It is 382

important to highlight that, in the opposite way, low FAEE concentrations may 383

not be indicative of high quality, since water addition during the extraction stage 384

diminishes the EtOH presence (Olimerca, 2015). 385

To sum up: the formation of FAEE depends, in addition on technological 386

aspects, on the occurrence of the corresponding substrates: free fatty acids and 387

short-chain (from 1 to 4 C-atom) alcohols, mainly EtOH. The FAEE 388

concentration is not something static. We have demonstrated that it increases 389

17

with time under certain storage conditions, going above the maximum allowed 390

limits in a few months. This evolution over time may lead, in the case of EVOO 391

obtained from healthy, mature (therefore, not necessarily overripe) fruits, to 392

FAEE concentrations above the limit permitted to classify the oils as extra 393

virgin, pushing them out of the highest category in a few-month time. This will 394

be accompanied by the consequent economic loss, being the most acute 395

difference between the extra virgin and the refined categories (International 396

Olive Council, 2015a). 397

To circumvent the presence of short-chain alcohols in the media once the oil 398

has been extracted, filtration before storage is recommended. 399

As previously observed (Pérez-Camino, Moreda, & Cert, 2001), controlled 400

temperature conditions (around 20 ºC) will decrease TAG hydrolysis, 401

diminishing substrate (free fatty acids) concentration. Besides, low temperature 402

will also prevent such substrate from reaching the activation energy needed to 403

turn it into product, therefore the importance of optimising the storage 404

conditions. 405

Finally, the economic crisis has pressed the first stages of the production 406

chain (olive milling), decreasing the prices that are being paid to the farmers 407

(International Olive Council, 2015b), who in turn may look for the maximum 408

yield harvesting at the latest (sometimes even overripe) stage of maturation. 409

Besides, olive oil is getting a low-price image and the market loss of 410

manufacturing brands is becoming very serious (International Olive Council, 411

2015b). Therefore, it may be positive to revise the current olive oil chain 412

situation together with the knowledge of the different parts involved regarding 413

the official limits –in the case at hand, FAEE maximum allowed concentration-, 414

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their obligatory nature, and the continuous implementation of good practices 415

focused on getting virgin olive oil of the maximum quality consistently, 416

economically and efficiently. 417

418

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420

Acknowledgements 421

The authors would like to thank Mss Diana Gómez Castillo for her technical 422

assistance and to the Coordination for the Improvement of Higher Education 423

Personnel (CAPES, Brazil) and National Council for Scientific and 424

Technological Development (CNPq, Brazil) for financial support (Brazilian 425

scholarship). 426

427

428

429

Conflict of Interest 430

The authors have declared no conflict of interest. 431

432

433

434

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applications. In R. Aparicio, & J. Harwood (Eds.), Handbook of olive oil. 437

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Dordrecht-London. 439

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Beltrán, G., Bejaoui, M. A., & Sánchez-Ortiz, A. (2015). Ethanol in olive fruit. 440

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European Community, L248, 1-102. 458

*European Commission Regulation (2011). EU No 61/2011 of 24 January 2011 459

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20

characteristics of olive oil and olive-residue oil and on the relevant methods 465

of analysis. Official Journal of the European Community, L338, 1-37. 466

Gómez-Coca, R. B., Cruz-Hidalgo, R., Fernandes, G. D., Pérez-Camino, M. C., 467

& Moreda, W. (2014). Analysis of methanol and ethanol in virgin olive oil. 468

MethodsX 1, e207-e211. 469

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alkyl esters presence in olive oil vs. organoleptic assessment. Food 471

Chemistry, 135, 1205-1209. 472

*International Olive Council (2010). Trade standard applying to olive oils and 473

olive pomace oils. COI/T. 15/NC No 3/Rev. 5, 1-19. 474

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monitoring of skilled virgin olive oil tasters. COI/T. 20/Doc. No 14/Rev. 3, 1-476

13. 477

International Olive Council (2011b). Sensory analysis of olive oil. Method for the 478

organoleptic assessment of virgin olive oil. COI/T. 20/Doc. No 15/Rev. 4, 1-479

14. 480

International Olive Council (2012). Determination of the content of waxes, fatty 481

acid methyl esters and fatty acid ethyl esters by capillary gas 482

chromatography using 3 grams of silica. COI/T. 20/Doc. No 31, 1-14. 483

*International Olive Council (2013a). Trade standard applying to olive oils and 484

olive-pomace oils. COI/T. 15/Doc. No 3/Rev. 7, 1-19. 485

International Olive Council (2013b). List of laboratories undertaking the sensory 486

analysis of virgin olive oils recognised by the International Olive Council for 487

the period from 1.12.2013 to 30.11.2014. T.28/Doc. No 3/Rev. 16, 1-15. 488

21

International Olive Council (2015a). 489

http://www.internationaloliveoil.org/estaticos/view/133-eu-producer-prices. 490

Uploaded on August 26th. 491

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http://www.internationaloliveoil.org/estaticos/view/307-the-olive-oil-value-493

chain-in-spain. Uploaded on August 26th. 494

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oli extra vergini di oliva [Possible increase of alkyl esters in extra virgin olive 496

oil] In Italian. La Rivista Italiana delle Sostanze Grasse, 88, 3-10. 497

Mariani, C., & Bellan, G. (2012). Sulla determinazione degli alcoli metilico ed 498

etilico negli oli extra vergini di oliva [Determination of methyl and ethyl 499

alcohols in olive oils] In Italian. La Rivista Italiana delle Sostanze Grasse, 39, 500

215-220. 501

Olimerca (2015) http://www.olimerca.com/noticiadet/ uploaded on May 13th. In 502

Spanish. 503

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W. (2008). Alkyl esters of fatty acids a useful tool to detect soft deodorized 505

olive oils. Journal of Agriculture and Food Chemistry, 56, 6740-6744. 506

Pérez-Camino, M. C., Moreda, W., & Cert, A. (2001). Effects of olive fruit quality 507

and oil storage practices on the diacylglycerol content of virgin olive oils. 508

Journal of Agriculture and Food Chemistry, 49, 699-704. 509

Pérez-Camino, M. C., Moreda, W., Mateos, R., & Cert, A. (2002). Determination 510

of esters of fatty acids with low molecular weight alcohols in olive oils. 511

Journal of Agriculture and Food Chemistry, 50, 4721-4725. 512

22

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in fruit ripening, enhancement of fruit quality and fruit deterioration. 514

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Saba, A., Mazzini, F., Riffaelli, A., Mattei, A., & Salvadori, P. (2005). 516

Identification of 9(E),11(E)-18:2 fatty acid methyl ester at trace level in 517

thermal stressed olive oils by GC coupled to acetonitrile CI-MS and CI-518

MS/MS, a possible marker for adulteration by addition of deodorized olive oil. 519

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Serani, A., & Piacenti, D. (2001). Sistema analitico per l’identificazione di oli 521

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vergini di oliva [Identification of deodorized oils in virgin olive oils. Note 1 – 523

Analysis of chlorophyll pigment in virgin olive oils] In Italian. La Rivista 524

Italiana delle Sostanze Grasse, 78, 459-463. 525

Serani, A., Piacenti, D., & Staiano, G. (2001). Sistema analitico per 526

l’identificazione di oli deodorati in oli vergini di oliva. Nota 2 - Cinetica di 527

isomerizzazione dei digliceridi in oli vergini di oliva [Identification of 528

deodorized oils in virgin olive oils. Note 2 - Kinetics of diacylglycerols 529

isomerization in virgin olive oils] In Italian. La Rivista Italiana delle Sostanze 530

Grasse, 78, 525-528. 531

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York-Heidelberg-Dordrecht-London. 534

535

536

23

Table 1 Fatty acid ethyl ester (FAEE) presence in the virgin olive oil under study 537

measured at different times. Comparison between the evolution at 20 ºC and at 538

40 ºC. Before spiking, the oil had been classified as extra virgin of the highest 539

quality according to the existing legislation. After spiking, the oil, divided into 540

three separated batches, had initial acidity values of 0.22 %, 0.38 %, and 0.77 541

%, respectively. The initial ethanol (EtOH) concentrations are given too. The 542

yellow highlighted figures indicate situations in which the oil is either near or 543

passed the critical 30 mg/kg value. Each value corresponds to the average of at 544

least two individual data. Three times the standard of the repeatability (3SDr) is 545

also given. 546

547

24

Figure captions 548

Fig. 1. Fatty acid alkyl ester (FAAE) presence in the virgin olive oil under study 549

measured at different times, obtained as the sum of the contents of the fatty 550

acid methyl esters (FAME) and the fatty acid ethyl esters (FAEE) from C16 to 551

C18 fatty acids. Comparison between the evolution at 20 ºC (dashed lines) and 552

at 40 ºC (solid lines). Before spiking, the oil had been classified as extra virgin 553

of the highest quality according to the existing legislation. After spiking, the oil 554

had an initial acidity value of A) 0.22 %, B) 0.38 %, and C) 0.77 %. The initial 555

methanol and ethanol concentrations, respectively, were: A) 42 and 68 mg/kg 556

(circles), 23 and 43 mg/kg (triangles), and 17 and 24 mg/kg (squares); B) 52 557

and 58 mg/kg (circles), 37 and 44 mg/kg (triangles), and 25 and 22 mg/kg 558

(squares); C) 86 and 55 mg/kg (circles), 25 and 47 mg/kg (triangles), and 27 559

and 20 mg/kg (squares) 560

561

25

Highlights 562

-Fatty acid ethyl ester concentration in virgin olive oil increases with time. 563

-Extra virgin olive oil may face a critical quality situation in a few-month time. 564

-Storage determines substrate availability for fatty acid ethyl ester formation. 565

-Poor virgin olive oil storage temperature favours fatty acid ethyl ester 566

formation. 567

26

Table 1 568

Acidity (initially) = 0.22 %

Initial [EtOH] = 24 mg.kg-1 Initial [EtOH] = 43 mg.kg-1 Initial [EtOH] = 68 mg.kg-1

Time, days

FAEE,

mg.kg

-1

40 ºC 3SDr

FAEE,

mg.kg

-1

20 ºC 3SDr

FAEE,

mg.kg

-1

40 ºC 3SDr

FAEE,

mg.kg

-1

20 ºC 3SDr

FAEE,

mg.kg

-1

40 ºC 3SDr

FAEE,

mg.kg

-1

20 ºC 3SDr

0 1.72 0.42

1.92 0.15 1.49 0.36 1.29 0.25

4 2.02 0.21 1.37 0.25

1.59 0.21

1.65 0.17

12

0.97 0.11 2.41 0.23 1.05 0.15

1.26 0.19

18 3.61 0.34 2.29 0.04 2.91 0.11 3.95 0.76 3.71 0.25 3.22 0.06

27 1.92 0.17

3.92 0.32 1.26 0.23 4.78 0.68 2.35 0.23

34 5.25 0.17

3.94 0.06 2.54 0.02 5.79 0.64 3.43 0.04

48 3.74 0.19 2.15 0.49 6.92 0.40 2.15 0.36 5.85 1.25 3.37 0.04

55 5.89 0.06

7.77 1.59 2.01 0.02 12.09 1.78 3.46 0.28

71 7.14 0.36 2.16 0.30 9.16 0.08 2.69 0.17 13.98 1.80 4.40 0.11

84 7.86 1.40 2.26 0.06 11.02 0.21 2.94 0.59

4.65 0.72

91 8.41 0.93 1.80 0.08 11.25 0.30 3.77 0.08 15.52 3.16

98 7.96 1.40 2.27 0.23 13.25 1.89 2.67 0.38 17.17 0.59 3.18 0.47

117 8.90 0.38 2.27 0.17 14.08 0.55 3.08 0.28 19.32 3.69 5.10 0.36

134 13.57 1.72 2.55 0.30 21.41 4.45 4.03 0.87 32.38 1.85 5.41 0.02

154 18.35 1.42 3.35 1.10 28.14 4.43 4.36 0.21 39.45 5.32 7.15 0.28

188 26.30 5.41 4.49 1.48 42.78 1.99 6.46 1.53 67.07 6.19 9.73 1.15

209 28.12 0.00 4.59 0.34 45.01 5.24 6.61 0.06

9.92 0.23

224 32.96 8.80 5.40 0.51 45.90 13.19 7.42 0.36 71.95 4.09 10.96 2.40

Acidity (initially) = 0.38 %

Initial [EtOH] = 22 mg.kg-1 Initial [EtOH] = 44 mg.kg-1 Initial [EtOH] = 58 mg.kg-1

0 2.38 0.34 1.94 0.36 3.23 0.56 3.66 0.21 2.38 0.51

4 2.89 0.81 2.03 0.36 4.16 0.67 2.49 0.36 2.98 0.23 2.47 0.36

12

3.01 0.53 9.83 0.04 3.29 0.38

4.15 0.02

18 4.26 0.89 4.26 0.02 6.65 0.41 4.76 0.81 7.35 0.62 4.38 0.91

27 6.08 1.04 2.20 0.28 8.28 1.55 4.05 0.00

4.50 0.17

34 8.40 1.57 5.00 0.70 11.50 0.19 4.89 0.78 10.55 1.36 5.43 0.21

48 9.08 1.85 5.03 0.62 13.21 0.45 7.29 0.83 21.38 1.06 6.12 0.06

27

Table 1 cont.

55 13.65 1.70 5.32 0.28 19.74 0.49 7.96 0.49 26.79 2.72

71 14.78 3.56

22.23 1.04 6.59 0.51 30.08 4.62 8.80 0.36

84 16.48 1.19 6.41 0.11 26.47 5.58 36.76 2.88 9.03 0.64

91 16.83 1.29 5.29 0.98 27.94 0.36 6.59 0.59 41.80 15.68 8.53 0.38

98 17.59 0.51 5.05 0.21 26.24 2.21 7.48 1.91 39.44 0.28 8.99 1.29

117 6.48 0.17 32.28 1.87 7.54 0.28 10.60 1.04

134 29.15 0.21 7.81 1.76 52.42 1.08 11.57 0.45 70.49 18.65 14.37 0.30

154 33.30 0.53 10.08 1.38 50.77 8.89 15.18 1.80 73.93 3.73 18.97 4.37

188 47.80 4.14 12.38 0.57 74.72 4.39 20.75 2.50 116.32 11.96 24.00 3.56

209 44.77 4.52 12.22 0.06 17.77 0.13 23.76 0.55

224 50.32 11.31 13.47 0.17 20.71 4.31 25.08 0.72

Acidity (initially) = 0.77 %

Initial [EtOH] = 20 mg.kg-1 Initial [EtOH] = 47 mg.kg-1 Initial [EtOH] = 55 mg.kg-1

0 2.49 0.23 3.03 0.11 2.42 0.69 3.14 0.49

4 3.14 0.47 2.51 0.57

3.31 0.25

3.19 1.00

12 7.55 1.63 3.33 0.25 11.09 4.24 3.26 0.02 6.36 2.08 3.38 1.17

18 4.45 1.02 2.96 0.21 8.57 0.78 5.21 0.98 9.68 0.04 2.92 0.28

27 6.26 1.91 3.04 0.19 11.49 1.12 5.57 1.04 13.87 1.06 5.40 0.53

34 10.33 1.48 4.89 1.06 16.63 4.79 7.45 1.42 20.62 2.38 5.77 1.17

48 16.26 0.78 5.07 1.15 27.78 2.08 8.73 0.59 28.41 2.35 8.58 0.42

55 18.32 2.78 6.55 0.08 38.77 3.71 11.04 0.47 34.33 4.09 9.05 0.02

71 5.59 0.21 46.25 7.81 12.81 1.57 52.40 2.46

84 21.70 0.06 6.97 0.86 47.13 8.38 12.83 2.25 48.49 1.99 15.03 0.11

91 23.54 0.23 6.76 0.74 47.61 3.33 11.82 1.29 50.76 8.59 15.74 1.29

98 20.26 1.59 6.56 0.25 51.71 18.63 13.35 1.97 48.41 2.27

117 22.72 9.76 6.78 0.95 50.77 0.11 14.54 0.32 55.57 0.21 14.41 0.95

134 37.62 0.62 10.29 1.10 88.20 4.43 23.02 4.14 99.75 20.60 17.33 1.72

154 38.01 2.31 14.00 3.50 95.11 0.98 29.42 4.01

26.79 4.43

188 51.43 5.54 18.21 0.23 38.06 2.44 34.02 3.25

209 16.08 1.46 35.44 0.68 44.92 1.70

224 17.42 3.82 36.43 4.35

28

Figure 1